Go from cell seeding to gene knockout in days
cat no | ioEA1094 Early Access
CRISPR-Ready
ioMicroglia
Male human iPSC donor-derived microglia expressing Cas9 for rapid gene knockout generation
CRISPR-Ready ioMicroglia are built from our well-established wild type ioMicroglia Male, engineered to constitutively express Cas9 nuclease. These cells arrive ready for guide RNA (gRNA) delivery from day 1 to 18 post-thaw. Using our optimised lentivirus or lipid-based gRNA delivery protocol, users can maximise their knockout efficiency and start measuring readouts from gene knockouts and CRISPR screens within days.
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Confidently investigate your phenotype of interest across multiple clones with our disease model clone panel. Detailed characterisation data (below) and bulk RNA sequencing data (upon request) help you select specific clones if required.
Benchtop benefits
Ready to use
Defined, characterised and functional human microglia constitutively expressing Cas9, ready for knockout experiments from day 1 to day 18 post-thaw.
Quick and easy
Generate readouts within days with a simple protocol for cell maturation and guide RNA delivery.
High knockout efficiency
Optimised protocols for lipid or lentivirus based guide RNA delivery to maximise knockout efficiency, as validated in a single gene knockout experiment and CRISPR screen.
Ready within days
Schematic overview of the timeline in the user manual
CRISPR-Ready ioMicroglia are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. The protocol for the generation of these cells is a three-phase process: an Induction phase that is carried out at bit.bio, Phase 1: Stabilisation for 24 hours, Phase 2: Maturation for a further 9 days, Phase 3: the Maintenance phase. Guide RNAs may be delivered between day 1 and 18 post-thaw and readouts may be performed 5 days after delivery. The detectability of a knockout at the protein level is influenced by both the chosen target and the half-life of the protein in question, and so the timepoint of the readout needs to be adjusted to the protein of interest.
Ready to generate gene knockouts
Flow cytometry analysis demonstrates high knockout efficiency of beta-2 microglobulin (B2M) by both lentiviral transduction and lipid-based transfection
Flow cytometry analysis of B2M protein expression in CRISPR-Ready ioMicroglia, after delivery of gRNA targeting B2M. gRNAs were introduced into the cells at day 10 post-thaw using two methods: lentiviral transduction or synthetic gRNA delivery with Lipofectamine RNAiMAX transfection reagent. After 5 days of culture following guide delivery, B2M gene knockout efficiency was assessed by flow cytometry analysis. (A) Lentiviral transduction with gRNA targeting B2M: 20% of cells received a B2M gRNA, as measured by GFP expression. A high knockout efficiency of 86% was achieved in these GFP+ cells (B). (C) Lipid-based transfection with gRNA targeting B2M: a high knockout efficiency of 82% was achieved.
Microglia at scale for CRISPR screens
A pooled single cell CRISPR knockout screen uncovers modulators of microglia activation
To conduct a pooled single cell CRISPR knockout screen (scCRISPR screen) with a targeted sequencing readout, we first identified a transcriptomic activation signature of 258 differentially expressed genes by comparing CRISPR-Ready ioMicroglia treated with and without LPS at day 10. Separately, we selected 110 candidate genes for the pooled scCRISPR screen based on their known roles in neurodegeneration and neuroinflammation. Guide RNAs were delivered via lentiviral transduction on day 10, aiming for a single integration per cell. The cells were cultured and then treated with +/- LPS for 24 hours before single cell processing on day 15. Cosine similarity analysis compared knockouts in LPS-treated CRISPR-Ready ioMicroglia to both resting and activated states. The analysis identified 17 gene knockouts that altered responses to LPS stimulation. The heatmap shows Log2FC profiles for gene knockouts that had a cosine similarity above 0.3 (arbitrarily chosen threshold) compared to cells with non-targeting guides in the unstimulated condition. Knockouts are sorted based on their cosine similarity to the non-LPS condition. CD14, MAP3K7, TIRAP, IKBKG, TRAF6, IKBKB, LY96, TICAM1, RELA, and TLR4 are genes known to be involved in LPS activation mediated via the TLR4 signalling pathway.
Highly characterised and defined
CRISPR-Ready ioMicroglia show ramified morphology by day 10
CRISPR-Ready ioMicroglia mature rapidly and key ramified morphology can be identified by day 4 and continues through to day 10, similarly to ioMicroglia Male (io1021). Day 1 to 10 post-thawing; 100x magnification.
CRISPR-Ready ioMicroglia express key microglia markers
Immunofluorescent staining on day 10 post-revival demonstrates similar homogenous expression of microglia markers P2RY12 and IBA1 and ramified morphology in CRISPR-Ready ioMicroglia compared to ioMicroglia Male (io1021). 100X magnification.
Key microglia functions
Phagocytosis of E. coli particles by CRISPR-Ready ioMicroglia
(A) Phagocytosis assay using pHrodo™ E. coli BioParticles™ at day 10 post-thaw demonstrates efficient uptake of bacteria particles by CRISPR-Ready ioMicroglia in comparison to ioMicroglia Male (io1021) after 4 hours of treatment. (B) An increase of fluorescence intensity of E.coli particles upon pH change in the phagosome, can be readily detected by fluorescence microscopy. A steep increase of fluorescence signal intensity was measured in the presence of E.coli particles alone, but not in combination with Cytochalasin D (CytoD), an inhibitor of actin polymerization.
CRISPR-Ready ioMicroglia secrete pro-inflammatory cytokines upon activation
CRISPR-Ready ioMicroglia were stimulated at day 10 post-thaw with LPS 100 ng/ml and IFNɣ 20 ng/ml for 24 hours. Supernatants were harvested and analysed by quantitative ELISA. These cells secrete the pro-inflammatory cytokines, TNF⍺ and IL-6 upon activation in comparison to ioMicroglia Male (io1021).
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Giving you access to endless and reliable human cells
“To do a genome-level CRISPR screen, with all the necessary replicates, requires billions of cells. Reaching that scale with iPSCs has been a significant challenge, so, many people turn to immortalised cell lines. But these cells are quite different from neurons in the human body. The development of ioCRISPR-Ready Cells is a huge step forward because it allows us to perform large-scale CRISPR screens on cells that closely resemble their in vivo counterparts—it’s a more physiologically relevant way of doing things.”
Emmanouil Metzakopian
Former Group leader, UK Dementia Research Institute, Cambridge University.
VP R&D, bit.bio.
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